Three-dimensional culture of primary cancer cells using tumor tissue

ABSTRACT

A method of producing a cell mass by three-dimensional culture of primary cancer cells having proliferative ability and properties of handleability, versatility, and high-throughput performance, in which a tumor tissue is used as a starting material, proliferation of cells such as fibroblasts other than cancer cells is inhibited, and the cell mass includes primary cancer cells as a main component. The object is achieved by providing a method of producing a cell mass by three-dimensional culture of primary cancer cells using a tumor tissue, including: a three-dimensional culture step of culturing cells obtained from the tumor tissue in a medium containing a 5% v/v or less extracellular matrix on a substantially low-adhesive cell culture substrate.

TECHNICAL FIELD

The present invention relates to a method of producing a cell mass bythree-dimensional culture of primary cancer cells using a tumor tissue,and a cell mass, a screening method, a determination method, and a kit.

BACKGROUND ART

Established cancer cells (cancer cell lines) have been conventionallyused for a method of evaluating tumor ex vivo. However, it has beenpointed out that cancer cell lines have lost the nature of theiroriginal tumor due to the homogenization of population and theaccumulation of gene mutations as a result of adaptation to an ex vivoenvironment with long time cell culture. In addition, it has been alsopointed out that the limited number of cancer cell lines cannot fullyexplain the pathological conditions of tumor composed of cancer cellswith diverse genetic heterogeneity. Therefore, in order to understand atumor more correctly, a culture system using primary cells of the tumorhas been gaining attention. In addition to a tumor obtained from apatient, a xenograft (patient-derived xenograft, PDX) tumor produced bygrafting the tumor to an immunodeficient animal is used.

Basic steps of culturing primary cells of a tumor include physically orenzymatically dispersing an extracted tumor and seeding the obtaineddispersed cells with a medium in a culture container, thereby allowingthe cells to proliferate in a CO₂ incubator (Non Patent Literature 1:Tissue Culture Technology-Basics-(Soshiki Boiyo no Gijutsu-Kisohen-),the 3rd edition, Asakura Publishing Co., Ltd., 1996). In order toimprove proliferation of cancer cells or inhibit excessive proliferationof non-cancer cells (especially fibroblasts), cell separation by densitygradient centrifugation, coating of a culture container with anextracellular matrix, use of a serum-free medium, cell separation basedon differences in sensitivity to trypsin enzymes and antibiotics, andthe like have been conducted. However, even with such creativetechniques, it is difficult to achieve cancer cell proliferation withhigh probability, and a more reliable culture method has been desired.

In recent years, a method of three-dimensionally culturing cells hasbeen gaining attention as a method of culturing primary cancer cells. Afirst example of such a method is the organoid culture method developedby Cleavers et al. from the Hubrecht Institute (Non Patent Literature 2:Sato, Toshiro, et al. “Single Lgr5 stern cells build crypt villusstructures in vitro without a mesenchymal niche,” Nature 459.7244(2009): 262-265; Non Patent Literature 3: Sato, Toshiro, et al.“Long-term expansion of epithelial organoids from human colon, adenoma,adenocarcinoma, and Barrett's epithelium,” Gastroenterolony 141.5(2011): 1762-1772). Organoid culture is a method of forming a cell mass(organoid) by self-organization of living stem cells. Specifically,living stem cells are embedded in extracellular matrix gel and culturedin an optimal medium. By applying this technique to primary cancercells, they have established a method of culturing large bowel cancer(Non Patent Literature 3), prostate cancer (Non Patent Literature 4:Ciao, Dong, et al. “Organoid cultures derived from patients withadvanced prostate cancer,” Cell 159.1 (2014): 176-187), and pancreaticcancer (Non Patent Literature 5: Boj, Sylvia F., et al. “Organoid modelsof human and mouse ductal pancreatic cancer,” Cell 160.1 (2015):324-338). However, this method requires embedding cells in gel at lowtemperatures, and thus, lacks high-throughput performance, and is notsufficiently versatile in drug development and the like.

A second example thereof is the 3D-tumor growth assay (3D-TGA) methoddeveloped by Molecular Response LLC and others. This method is similarto the first method in that cells are embedded in extracellular matrixgel and cultured using an optimal medium, but is different in thatpreculture is performed in a culture container coated with anextracellular matrix in the prior step, and grown cancer-associatedfibroblasts (CAFs) or mesenchymal stem cells (MSCs) are optionallyembedded with cancer cells in extracellular matrix gel (Non PatentLiterature 6: Saunders, John H., et al. “Individual patient oesophagealcancer 3D models for tailored treatment,” Oncotarget (2016)). Thismethod also requires embedding cells in gel at low temperatures as inthe first method, and thus, lacks high-throughput performance.

A third example thereof is the culture method using a non-uniformlydispersed cell mass having a diameter of from 40 to 100 μm (cancertissue-originated spheroid, CTOS) developed by Inoue et al. from theCenter for Adult Diseases (the CTOS method) (Non Patent Literature 7:Kondo, Jumpei, et al. “Retaining cell-cell contact enables preparationand culture of spheroids composed of pure primary cancer cells fromcolorectal cancer” Proceedings of the National Academy of Sciences108.15 (2011): 6235-6240). Specifically, the obtained CTOSs are seededon a non-adhesive plate and culture is conducted while suspending theCTOSs using an optimal medium. As cancer cells of CTOSs can proliferatewithout being embedded in extracellular matrix gel, this method does notrequire a temperature control operation as compared with the first andsecond methods. However, as the drug sensitivity test requires acomplicated step of selecting and arranging CTOSs of a uniform size,high-throughput performance is poor and cancer cells have poorproliferative ability in the method as compared to the method in whichcells are embedded in an extracellular matrix. Therefore, the method isinsufficient in terms of practical use.

A fourth example thereof is the culture method using a cell cultureplate treated for inhibiting adhesion to uniformly dispersed cancercells developed by Nakatsura et al. from the National Cancer Center(Patent Literature 1: WO2016/047801). Specifically, it is a method inwhich uniformly dispersed cancer cells are seeded with a mediumcontaining serum at 1% by volume or more on a NanoCulture Plate, whichis a three-dimensional culture plate manufactured by ORGANOGENIX, Inc.so as to culture a cell mass. A culture kit (Cancer Organoid CultureKit) is commercially available from ORGANOGENIX, Inc. As cancer cellscan proliferate without being embedded in extracellular matrix gel, thismethod does not require a temperature control operation as compared withthe first and second methods. In addition, since uniformly dispersedcancer cells are used, it is possible to uniformly seed cells withoutthe need for special operations, the method is superior to the thirdmethod in terms of high-throughput performance. However, there are onlyreports using a human lung cancer tumor and a xenograft tumor of breastcancer (Non Patent Literature 8: Sakamoto. Ruriko, et al. “Time-lapseimaging assay using the BioStation CT: A sensitive drug-screening methodfor three-dimensional cell culture,” Cancer science 106.6 (2015):757-765). In particular, in the xenograft tumor of breast cancer,proliferation of cell mass could not be clearly confirmed, verificationof versatility has remained unsatisfactory, and there is a questionabout practical use.

As described above, in culture of primary cancer cells using a tumortissue, the certainty of culture performance is increasing because ofthe use of the three-dimensional culture methods. However, each methodis problematic in terms of proliferative ability, handleability,high-throughput performance, versatility, etc. of cancer cells.

CITATION LIST Patent Literature

Patent Literature 1: WO2016/047801

Non Patent Literature

Non Patent Literature 1: Tissue Culture Technology-Basics-(Soshiko Baiyono Gijustu-Kisohen-), the 3rd edition, Asakura Publishing Co., Ltd.,1996

Non Patent Literature 2: Sato, Toshiro, et al, “Single Lgr5 stem cellsbuild crypt villus structures in vitro without a mesenchymal niche.”Nature 459.7244 (2009): 262-265

Non Patent Literature 3: Sato, Toshiro, et al, “Long-term expansion ofepithelial organoids from human colon, adenorna, adenocarcinoma, andBarrett's epithelium.” Gastroenterology 141.5 (2011): 1762-1772

Non Patent Literature 4: Gao, Dong, et al, “Organoid cultures derivedfrom patients with advanced prostate cancer.” Cell 159.1 (2014): 176-187

Non Patent Literature 5: Boj, Sylvia F., et al. “Organoid models ofhuman and mouse ductal pancreatic cancer.” Cell 160.1 (2015): 324-338

Non Patent Literature 6: Saunders, John H., et al. “Individual patientoesophageal cancer 3D models for tailored treatment.” Oncotarget (2016)

Non Patent Literature 7: Kondo, Jumpei, et al. “Retaining cell-cellcontact enables preparation and culture of spheroids composed of pureprimary cancer cells from colorectal cancer.” Proceedings of theNational Academy of Sciences 108.15 (2011): 6235-6240

Non Patent Literature 8: Sakamoto, Ruriko, et al. “Time-lapse imagingassay using the BioStation CT: A sensitive drug-screening method forthree-dimensional cell culture.” Cancer science 106.6 (2015): 757-765

SUMMARY OF INVENTION

The invention has been made in view of the above problems. An object ofthe invention made by the present inventors is to provide a method ofproducing a cell mass by three-dimensional culture of primary cancercells having proliferative ability and properties of handleability,versatility, and high-throughput performance, in which a tumor tissue isused as a starting material, proliferation of cells such as fibroblastsother cancer cells is inhibited, and the cell mass includes primarycancer cells as a main component.

In order to solve the above problems, the inventors made intensivestudies. As a result, they found that it is possible to produce a cellmass of primary cancer cells having high proliferative ability andproperties of handleability, versatility, and high-throughputperformance of cancer cells that can be used in various tests, by athree-dimensional culture method by culturing uniformly dispersed cellsin suspension using a patient-derived xenograft (hereinafter alsoabbreviated as “PDX” in some cases) tumor as a starting material bycombining an optimal culture substrate and an optimal three-dimensionalculture medium. This has led to the completion of the invention.

The invention is exemplified as follows.

-   [1] A method of producing a cell mass by three-dimensional culture    of primary cancer cells using a tumor tissue, comprising:

a three-dimensional culture step of culturing cells obtained from thetumor tissue in a medium containing a 5% v/v or less extracellularmatrix on a substantially low-adhesive cell culture substrate.

-   [2] The method according to [1], wherein the tumor tissue is a    xenograft tumor.-   [3] A cell mass of primary cancer cells obtained from a tumor    tissue, which is produced by the method of producing a cell mass    according to [1] or [2].-   [4] A method of screening for a substance that acts on a cell mass,    comprising:

producing a cell mass of primary cancer cells by the method of producinga cell mass according to [1] or [2];

adding a test substance to the cell mass; and

evaluating an action of the test substance on the cell mass.

-   [5] A method of determining effects of a substance on a cell mass,    comprising:

producing cell mass of primary cancer cells by the method of producingcell mass according to [1] or [2];

adding a test substance to the cell mass; and

evaluating effects of the test substance on the cell mass.

-   [6] A kit of producing a cell mass by three-dimensional culture of    primary cancer cells using a tumor tissue, including:

a substantially low-adhesive cell culture substrate; and

a medium containing a 5% v/v or less extracellular matrix for athree-dimensional culture step.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention has made it possible to provide a method of producing acell mass by three-dimensional culture of primary cancer cells havingproliferative ability and properties of handleability, versatility, andhigh-throughput performance, in which a tumor tissue is used as astarting material, proliferation of cells such as fibroblasts other thancancer cells is inhibited, and the cell mass includes primary cancercells as a main component.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of culture in Example 2 (image): *1: a PDXtumor of pancreatic cancer (1) was used; a culture medium prepared byadding Coming Matrigel GFR to SternPro hESC SFM to yield a finalconcentration of 2% v/v was used; *2: in a case in which spindle-shapedcells were observed on the bottom of the plate, it was determined thatthere was adhesion of fibroblasts.

FIG. 2 shows the results of culture in Example 3 (image): *1: a PDXtumor of pancreatic cancer (1) was used; PrimeSurface was used; *2: itwas identified as “impossible” in a case where cells or cell mass wasalso suctioned upon suctioning half of the medium in a well bypipetting.

FIG. 3-1 shows the results of culture (Coming Matrigel) in Example 4(image): *1: a PDX tumor of pancreatic cancer (1) was used; PrimeSurfacewas used; *2: it was identified as “impossible” in a case where cells orcell mass was also suctioned upon suctioning half of the medium in awell by pipetting.

FIG. 3-2 shows the results of culture (Cultrex BME) in Example 4(image): *1: a PDX tumor of pancreatic cancer (1) was used; PrimeSurfacewas used; *2: it was identified as “impossible” in a case where cells orcell mass was also suctioned upon suctioning half of the medium in awell by pipetting.

FIG. 3-3 shows the results of culture (Cultrex RGF BME) in Example 4(image): *1: a PDX tumor of pancreatic cancer (1) was used; PrimeSurfacewas used; *2: it was identified as “impossible” in a case where cells orcell mass was also suctioned upon suctioning half of the medium in awell by pipetting.

FIG. 3-4 shows the results of culture (Cellmatrix Type I-A) in Example 4(image): *1: a PDX tumor of pancreatic cancer (1) was used; PrimeSurfacewas used; *2: v/v wells were visually observed on Day 14 becausefocusing was not achieved in microscopy; *3: it was identified as“impossible” in a case where cells or cell mass was also suctioned uponsuctioning half of the medium in a well by pi petting.

FIG. 4 shows the results of culture in Example 5 (image): *1: a PDXtumor of pancreatic cancer (1) was used; a culture medium prepared byadding Coming Matrigel GFR to minimal essential medium to yield a finalconcentration of 2% v/v was used; PrimeSurface was used as a cultureplate.

FIG. 5 shows the results of culture in Example 6 (image): *1: a culturemedium prepared by adding Corning Matrigel GFR to StemPro hESC SFM toyield a final concentration of 2% v/v was used; PritneSurface was usedas a culture plate;

FIG. 6 shows the results of culture in Example 7 (image): *1: a culturemedium prepared by adding Coming Matrigel GFR to StemPro hESC SFM toyield a final concentration of 2% v/v was used; PrimeSurface was used asa culture plate; a cell mass of Day 14 was used.

FIG. 7 shows the results of culture in Example 8 (image): *1: a culturemedium prepared by adding Coming Matrigel GFR to StemPro hESC SFM toyield a final concentration of 2% v/v was used; PrimeSurface was used asa culture plate.

FIG. 8 shows the results of culture in Example 9 (image): *1: a culturemedium prepared by adding Coming Matrigel GFR to StemPro hESC SFM toyield a final concentration of 2% v/v was used; PrimeSurface was used asa culture plate. A half of medium exchange was performed with a culturemedium supplemented with gemcitabine on Day 7. A half of medium exchangewas performed with a culture medium supplemented with gemcitabine whichwas adjusted to maintain the final concentration on Days 10 and 12. OnDay 14, ATP assay was performed, and the proportion based on the resultof each gemcitabine concentration group was calculated when the resultat a gemcitabine concentration of 0 μmol/L was defined as a cellviability of 100%, thereby creating a cell viability curve.

FIG. 9 shows the results of culture in Example 10 (image).

FIG. 10-1 shows the results of culture in Example 11 (image).

FIG. 10-2 shows the results of culture in Example 11 (image).

DESCRIPTION OF EMBODIMENTS

The method of producing a cell mass by three-dimensional culture ofprimary cancer cells using a tumor tissue of the invention ischaracterized by a three-dimensional culture step of culturing primarycancer cells obtained from the tumor tissue in a medium containing a 5%v/v or less extracellular matrix on a substantially low-adhesive cellculture substrate.

In general, it was considered that when primary cancer cells aresubjected to three-dimensional culture in suspension, it is difficult toform a cell mass unless a special technique is used. In addition, it wasconsidered that when using a medium containing an extracellular matrix,culture in a gel-like medium in a fixed state results in a cell masshaving high proliferative ability. However, as mentioned in Examplesdescribed later, by culturing primary cancer cells in suspension in amedium containing a sol of an extracellular matrix on a substantiallylow-adhesive cell culture substrate, it has become possible to carry outthree-dimensional culture while inhibiting proliferation of cells suchas fibroblasts other than cancer cells, thereby allowing the primarycancer cells to have high proliferative ability. This has providedunexpected effects.

Accordingly, it is possible to produce a cell mass by three-dimensionalculture of primary cancer cells using a tumor tissue. The cell mass hasproperties of handleability, versatility, and high-throughputperformance of cancer cells because cells are cultured in suspensionwhile maintaining high proliferative ability suited for various tests.The suspended state means a state in which cells can be easily moved byan operation such as pipetting, but are not adhering to a cell culturesubstrate or embedded in a gel-type culture solution. The size of cellmass produced by the method of the invention is not particularlylimited. However, for example, the average diameter is 100 μm or more,preferably from 100 μm to 300 μm.

Hereinafter, one aspect of the method of producing a cell mass bythree-dimensional culture of primary cancer cells using a tumor tissuebased on a culture method according to the invention will be described.

One example of a substantially low-adhesive cell culture substrate thatcan be used in the invention is a low-adhesive culture substrate thatenables three-dimensional suspension culture of cells. Such asubstantially low-adhesive cell culture substrate may be a cell culturesubstrate which is made entirely low-adhesive so as to be used inthree-dimensional culture, and can be, for example, a culture substratehaving a hydrophilic surface or a cell culture substrate having asurface treated with a hydrophilic compound. Specific examples thereofinclude, for example, PrimeSurface (Sumitomo Bakelite Co., Ltd.),Corning ULA Round-bottom (Coming Incorporated), Corning ULA Flat-bottom(Coming Incorporated), and Elplasia (Kuraray Co., Ltd.). In addition, itis also possible to form a low-adhesive culture substrate by treating anadhesive cell culture substrate such that adhesiveness is inhibited. Asthe treatment for inhibiting adhesiveness, known treatment such ashydrophilization treatmentor hydrophobization treatment can be used. Inaddition, it is possible to determine whether a culture substrate islow-adhesive by performing three-dimensional culture of primary cancercells using a tumor tissue and confirming that cells such as fibroblastsother than cancer cells do not extend and proliferate in areas otherthan a cell mass on the substrate. In addition, the shape, processing,material, etc. of the substrate are not limited as long as it is asubstantially low-adhesive cell culture substrate.

The tumor tissue that can be used in the invention may he a tissuefragment containing known cancer cells. For example, cancer cells oflymphoma, myeloma, brain tumor, breast cancer, endometrial cancer,cervical cancer, ovarian cancer, esophageal cancer, gastric cancer,appendix cancer, large bowel cancer, hepatocellular carcinoma,gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenalcancer, gastrointestinal stromal tumor, mesothelioma, laryngeal cancer,oral cavity cancer, gingival cancer, tongue cancer, buccal mucosacancer, salivary adenocarcinoma, carcinoma of the paranasal sinuses,maxillary cancer, carcinoma of the frontal sinus, ethmoid cancer,carcinoma of the sphenoidal sinus, thyroid cancer, renal cancer, lungcancer, osteosarcoma, prostate cancer, testicular tumor, renal cellcarcinoma, bladder cancer, rhabdomyosarcoma, skin cancer, anal cancer,and other various cancers, various stem cells, various progenitor cells,mesenchymal progenitor cells, ES cells, and iPS cells can be mentioned.Cells are not limited to a single type of cells, but may be a populationof cells of different types. The origin of a tumor tissue is notparticularly limited. Examples thereof include animals belonging toprimates including humans and monkeys, animals belonging to rodents suchas mice and rats, animals belonging to the order Lagomorpha, animalsbelonging to the order Carnivora such as dogs and cats, animalsbelonging to the order Artiodactyla such as pigs, and animals belongingto the order Perissodactyla such as bovines and horses. In addition to atumor tissue obtained from a patient, a xenograft (patient-derivedxenograft, PDX) tumor produced by grafting the tumor tissue to animmunodeficient animal can also be used. The patient-derived xenografttumor can be prepared by making use of a known method (Non PatentLiterature 9: Cho, Sung-Yup, et al. “An integrative approach toprecision cancer medicine using patient-derived xenografts,” Moleculesand cells 39.2 (2016): 77).

According to the invention, even with the use of a PDX tumor, it ispossible to produce a cell mass by three-dimensional culture of primarycancer cells having high proliferative ability and properties ofhandleability, versatility, and high-throughput performance which can beused in various tests. It is therefore particularly preferable that aPDX tumor is a tumor of interest. PDX tumors enable the development ofanticancer drugs with high clinical predictability using patient-derivedtumors, and can be used also for determination of therapeutic effects.It is therefore particularly preferable that a PDX tumor can be a tumorof interest.

In addition, it is possible to use, as a tumor tissue that can be usedin the invention, either a fresh tumor prepared by surgically extractinga tumor and immersing it in a tissue preservation solution (such asphysiological saline or HBSS) or a cryopreserved tumor prepared byimmersing a fresh tumor in a cryopreservation solution (such asCELLBANKER 1) and cryopreserving it while keeping cells alive. A personskilled in the art can select a preservation method from known methodsas appropriate and use the method.

A known method can be used as a three-dimensional culture method thatcan be used in the invention unless otherwise specified.

A known method can be adopted as a step of preparing primary cancercells in a method of producing a cell mass by three-dimensional cultureof primary cancer cells using a tumor tissue that can be used in theinvention. For example, a method in which cells are collected from atissue fragment containing cancer cells or a method in which the tissuefragment is directly used can be mentioned. However, in consideration ofhandleability, test repeatability, and the like, the method in whichcells are collected is preferable.

In the method in which cells are collected from a tissue fragmentcontaining cancer cells, for example, a tumor tissue fragment extractedfrom the living body is treated by enzyme treatment, density gradientcentrifugation treatment, filter treatment, treatment using magneticbeads or a flow cytometer, or the like as necessary for separation andpurification. Enzyme treatment is preferable because the treatmentmethod is convenient such that cancer cells dispersed as single cellscan be easily obtained. The resulting cell group may be a population ofcells in different stages of differentiation originating from the sametissue.

The three-dimensional culture method that can be used in the inventionincludes culturing primary cancer cells obtained from a tumor tissue ina medium containing a 5% v/v or less extracellular matrix on theabove-described substantially low-adhesive cell culture substrate. Theextracellular matrix, the medium, and the primary cancer cells may bemixed in any order thereof. For example, a medium containing the 5% v/vor less extracellular matrix in a sol state is prepared and mixed withthe primary cancer cells, and the mixture is seeded on the substantiallylow-adhesive culture plate. In the case of using the 5% v/v or lessextracellular matrix, the primary cancer cells can be cultured insuspension.

Since the cells can be cultured in suspension, handleability of thecells is easy, the cells can be readily used in various tests, and amechanical operation of the cells becomes possible. Thus, the cells canbe readily used in high-throughput tests, which is preferable.

One example of an extracellular matrix that can be used in the inventionincludes an extracellular matrix that can be used in a knownthree-dimensional culture method. Examples thereof include, for example,collagen I, collagen IV, fibronectin, laminin, vitronectin, entactin,gelatin, elastin, proteoglycan, glucosaminoglycan, chondroitin sulfate,dermatan sulfate, heparan sulfate, heparin, keratan sulfate, Matrigel(trademark: Coming Incorporated), Matrigel GFR (trademark: ComingIncorporated), Cultrex BME (trademark: Trevigen, Inc.), Cultrex RGF BME(trademark: Trevigen, Inc.), Cellmatrix Type I-A (trademark: NittaGelatin Inc.), and growth factors (such as basic FGF, EGF, IGF-1, PDGF,NGF, and TGF beta)

The concentration of an extracellular matrix that can be used in theinvention can be set such that primary cancer cells obtained from atumor tissue can be three-dimensionally cultured in suspension. It maybe 5% v/v or less and 2.5% v/v or less. Although the lower limit thereofis not particularly limited, it is preferably 0.1% v/v or more, morepreferably 0.2% v/v or more, and particularly preferably 0.5% v/v ormore. A person skilled in the art can set the concentration asappropriated depending on the type of the primary cancer cells, etc.

The density of cells to be seeded that can be used in the invention canbe set such that primary cancer cells obtained from a tumor tissue cannormally live in the form of cell mass produced by a three-dimensionalculture method. The primary cancer cells can be seeded at a cell densityof usually from 3×10³ to 7×10⁴ cells/cm². However, it is possible to seta preferable cell density as appropriate depending on culture conditionsor culture instruments to be used. In addition, known conditions can beused as culture conditions. For example, the culture temperature ispreferably from 20° C., to 45° C., more preferably from 30° C. to 42°C., and particularly preferably from 35° C. to 39° C., and the pH of aculture solution is preferably pH 7 to 8. In addition, the cultureperiod can be set as appropriate depending on a test method of interest.However, it is preferably from 2 days to 30 days and more preferablyfrom 7 days to 14 days. The cell density can be stably maintained athigh levels even in a particularly long period of time (about 10 days ormore), resulting in a high value of use.

As a medium that can be used in the invention, a cell culture basemedium, a differentiation medium, a medium specialized for primaryculture, or the like can be optionally used. Examples thereof include,for example, Dulbecco's Modified Eagle Medium (DMEM), Glasgow's MEM(GMEM), RPM11640, Ham's F12 medium, and serum-free medium (such as MCDBmedium). Further, a medium prepared by adding serum, various growthfactors (insulin, transferrin, selenium salt, and dexamethasone), and adifferentiation inducer to any of these media can be used.

A cell mass that can be produced by three-dimensional culture of primarycancer cells using a tumor tissue according to the invention can be usedin various test methods, for example but not limited to, a method ofscreening for a substance that acts on the cell mass and a method ofdetermining effects of a substance on the cell mass. The cell mass hashigh cell proliferation ability that can be utilized in various testmethods and also has handleability, versatility, and high-throughputperformance because it exists in suspension, and thus, the cell mass isadvantageous in that it can be readily used in various tests.

A person skilled in the art can carry out a method of screening for asubstance that acts on a cell mass according to the invention bymodifying a known method as appropriate. For example, in accordance withthe invention, a method of screening for a substance that acts on a cellmass includes: a step of producing a cell mass by three-dimensionalculture of primary cancer cells using a tumor tissue; a step of adding atest substance to the cell mass; and a step of evaluating an action ofthe test substance on the cell mass. Examples of a substance that actson a cell mass include substances capable of directly and/or indirectlyacting on the cell mass such as, for example, anticancer drugs, variouscompounds, antibodies, antibody-drug conjugates, nucleic acids,peptides, viruses, and cells (such as NK cells, TCR-T cells, and CAR-Tcells). For example, as it is possible to screening for a substancecapable of inhibiting proliferation of the cell mass, the substance canbe used for the development of anticancer drugs.

A person skilled in the art can carry out a method of determiningeffects of a substance on the cell mass of the invention by modifying aknown method as appropriate. For example, in accordance with theinvention, a method of determining effects of a substance on a cell massincludes: a step of producing a cell mass by three-dimensional cultureof primary cancer cells using a tumor tissue; a step of adding a testsubstance to the cell mass; and a step of evaluating effects of the testsubstance on the cell mass. Examples of a test substance for determiningthe effects include, for example, anticancer drugs, various compounds,antibodies, antibody-drug conjugates, nucleic acids, peptides, viruses,and cells (such as NK cells, TCR-T cells, and CAR-T cells). For example,by identifying an anticancer drug capable of inhibiting proliferation ofthe cell mass from among various anticancer drugs and administering theanticancer drug to a patient as the origin of the cell mass, it ispossible to enhance treatment effects. This can support selection of atreatment method. It is also possible to use the anticancer drug to apatient having a similar origin to the cell mass without limiting to acombination of the cell mass and the patient as the origin of the cellmass.

Known anticancer drugs can be used. Examples thereof include, forexample, actinomycin D, melphalan, busulfan, carboplatin, cisplatin,cyclophosphamide, dacarbazine, oxaliplatin, procarbazine, temozolomide,ifosfamide, liposomal doxorubicin, doxorubicin, daunorubicin,epirubicin, idarubicin, mitomycin C, bleomycin, mitoxantrone,cladribine, fluorouracil, mercaptopurine, pemetrexed, methotrexate,cytarabine, nelarabine, capecitabine, fludarabine, gemcitabine,pentostatin, vincristine, eribulin, paclitaxel, vinblastine, irinotecan,docetaxel, etoposide, vinorelbine, nogitecan, paditaxel, tretinoin,bevacizumab, trastuzumab, panitumumab, cetuximab, ibritumomab tiuxetan,rituximab, gem:tuzuniab ozogamicin, everolimus, erlotinib, lapatinib,gefitinib, imatinib, dasatinib, sunitinib, sorafenib, bortezomib,tamibarotene, nimustine, ranimustine, enocitabine, carmofur, cytarabineocfosphate, tegafur, tegafur-uracil, tegafur-gimeracil-oteracilpotassium, doxifluridine, hydroxycarbamide, sobuzoxane, vindesine,aclarubicin, amrubicin, zinostatin stimalamer, pirarubicin, peplomycin,and nedaplatin.

In addition, the cell mass may be directly used in the culture solutionfor various tests or transferred to another container for used invarious tests after being cultured by the three-dimensional culturemethod. In a case in which the cell mass is transferred to anothercontainer, the cell mass can be collected by a known method. A personskilled in the art can select carry out the method as appropriate. Inaddition, the cell mass is advantageous in that it can be readilycollected because it is cultured in suspension.

Examples of various test methods include known test methods such as, forexample, cell proliferation tests (MIT assay, ATP assay, etc.),viable/dead cell staining analysis, phenotype screening (for example,analysis of epithelial-mesenchymal transition such as cell morphologychange), histopathological analysis (HE staining, immunohistochemicalstaining, etc.), and biochemical analysis (gene mutation analysis, mRNAexpression analysis, protein expression analysis, exosome analysis,etc.). A person skilled in the art can predetermine a test method asappropriate depending on a method of screening for the above-describedsubstance, a method of determining effects of the substance, and thelike.

According to the invention, it becomes possible to perform various testmethods using an ordinary automatic dispenser in seeding of cells on a96-well or 384-well plate, medium exchange, addition of a drug solution,addition of an assay reagent, acquisition of electronic data on thesubsequent luminescence or using a fluorescence measurement device,acquisition of electronic data on image analysis using an automaticimage analyzer, or the like in a convenient manner or in ahigh-throughput manner.

A person skilled in the art can readily confirm by a known method thatcell mass produced by three-dimensional culture of primary cancer cellsusing a tumor tissue according to the invention inhibits proliferationof cells such as fibroblasts other than cancer cells and has highproliferative ability because of containing primary cancer cells as amain component so that the cell mass reflects the living body, andtherefore, the cell mass can be used for various applications. Forexample, it is possible to confirm whether a cell mass has a functionsimilar to a tumor in the living body by visually observing cell massformation, evaluating proliferative ability of cells, evaluating effectsof a known substance on the cell mass, or evaluating whether the cellmass can form a tumor when the cell mass obtained from an animal isregrafted to the animal.

As the kit of the invention, a kit of producing a cell mass bythree-dimensional culture of primary cancer cells using a tumor tissue,which includes a substantially low-adhesive cell culture substrate and amedium containing a 5% v/v or less extracellular matrix for thethree-dimensional culture step, can be mentioned. Regarding the mediumcontaining a 5% v/v or less extracellular matrix for thethree-dimensional culture step, an extracellular matrix and a medium maybe separately provided so as to be mixed before use of the kit or anextracellular matrix and a medium may be mixed in advance and thenprovided. In addition, manufacturer's instructions, etc. contain thedescription of a substantially low-adhesive cell culture substrate, anextracellular matrix, and a medium that can be used in the method ofproducing a cell mass of the invention such that a user of the kit canobtain the kit components. In addition, for example, materials for acell culture substrate, a medium, an extracellular matrix, and the likeor a method of using the kit which can be used in the method ofproducing a cell mass of the invention can be mentioned.

EXAMPLES

Hereinafter, the invention will be described in more detail by way ofexamples. However, the invention is not limited by these examples.

Example 1: Collection of Tumor Tissue and Cancer Cell DispersionTreatment

A human cancer patient-derived xenograft (hereinafter referred to as“PDX”) tumor, which was subcutaneously grown in an immunodeficient mouse[super SCID mouse (strain name: C3H/HeJ/NOs-scid; LPS-nonresponder)],was aseptically extracted in a safety cabinet according to an ordinarymethod, and every necrotic area of the tumor was removed with surgicalscissors. The tumor was immediately immersed in a Japanese Pharmacopoeiasaline solution and preserved on ice. Next, the Japanese Pharmacopoeiasaline solution was removed from the tumor, and the tumor was washedthree times repeatedly with specimen treatment solution (included in aCancer Organoid Culture Kit, ORGANOGENIX, Inc.).

Cancer cell dispersion treatment was performed as described below as apreparation step for three-dimensional culture. The washed tumor wasplaced in a 10-cm petri dish on ice, shredded to a size of about 1 mmsquare with surgical scissors, and collected in a 50-mL tube. Adispersion solution (included in a Cancer Organoid Culture Kit,ORGANOGENIX, Inc) was added to the tube Tumor fragments wereenzymatically treated at 37° C. for 60 min while the tube was shaken ina water bath. The reaction was weakened by adding twice the amount of aspecimen treatment solution to the reaction solution. The undispersedresidues were removed by passing the mixture through a 100-μm cellstrainer. The tube and the cell strainer were washed with an appropriateamount of the specimen treatment solution, and cells were collected andcentrifuged at 300×g for 5 min. The supernatant was removed. Then, thespecimen treatment solution was added to resuspend the resulting cellpellet and centrifuged at 300×g for 5 min. Thereafter, the cell pelletwas resuspended with an appropriate amount of the specimen treatmentsolution and cell counting was performed. After confirming that thecells were separated into single cells, the cells were used in thefollowing experiment.

Example 2: Seeding and Culture of Cells in Three-Dimensional CulturePlate

At first, it was examined whether it would be possible to performthree-dimensional culture using a PDX tumor by means of a CancerOrganoid Culture Kit (ORGANOGENIX, Inc.) described as capable ofthree-dimensional culture of primary cancer cells. A Cancer OrganoidCulture Kit is for a method of performing culture with a plate preparedby allowing a low-adhesive plate to have an uneven scaffolding structurefor forming a cell mass of cancer cells and a medium containing serum at1% by volume or more.

Primary cancer cells were prepared using PDX tumor of pancreatic cancer(1) (procured from National institutes of Biomedical Innovation, Healthand Nutrition) in accordance with Example 1. The necessary amount ofcells counted after dispersion treatment were collected in a 15-mL tube,and the supernatant was removed by centrifugation at 300×g for 5 min.Thereafter, a cell suspension was prepared using NanoCulture Medium Ptype (included in a Cancer Organoid Culture ORGANOGENIX, Inc. so thatthe cell count was 1×10⁵ cells/mL. NanoCulture Medium P type in anamount of 150 μL was added to NanoCulture Plate (included in a CancerOrganoid Culture Kit, ORGANOGENIX, Inc.) serving as a three-dimensionalculture plate, followed by centrifugation at 700×g for 5 min. The platewas left to stand at 37° C. for 10 min for prewetting. The cellsuspension in an amount of 100 μL was added to the plate, and staticculture was initiated in a CO₂ incubator set to 37° C. and 5% CO₂. Theseeded cell count was 1×10⁴ cells/250 μL/well, and the day of seedingwas determined to he Day 0. A half of medium exchange was performed asappropriate. As a result, no clear cell mass was formed and adhesion ofspindle-shaped fibroblasts to the plate bottom was observed. It wastherefore found difficult to apply a Cancer Organoid Culture Kit ofORGANOGENIX, Inc. directly to the PDX tumor of pancreatic cancer (1).

In order to achieve culture in suspension, an extracellular matrix,which is usually used in the gel form, was prepared to be contained in amedium in a sol state, and it was examined whether it would be possiblefor primary cancer cells to form a cell mass without expansion orproliferation of cells such as fibroblasts other than cancer cells.

As described above, the necessary amount of cells counted were collectedin a 15-mL tube, and the supernatant was removed by centrifugation at300×g for 5 min. Thereafter, a cell suspension was prepared so that thecell count was 5×10⁴ cells/mL using a medium prepared by adding CorningMatrigel GFR (Corning Incorporated) to StemPro hESC SFM (Thermo FisherScientific K.K.) to yield a final concentration of 2% v/v. The cellsuspension in an amount of 200 μL was seeded in PrimeSurface (SumitomoBakelite Co., Ltd.), Corning ULA Round-bottom (Corning Incorporated),Coming ULA Flat-bottom (Coming Incorporated), or Elplasia (Kuraray Co.,Ltd.), which is a three-dimensional culture plate as an ordinarylow-adhesive plate, or a 96-well microplate (Coming Incorporated), whichis an ordinary adhesive plane (two-dimensional) culture plate, andstatic culture was initiated in a CO₂ incubator set to 37° C. and 5%CO₂. Similarly, a cell suspension was prepared to yield 1×10⁵ cells/mLin NanoCulture Plate, 100 μL of the cell suspension was added to theplate prewetted with 150 μL of a medium for seeding, and static culturewas initiated in a CO₂ incubator set to 37° C. and 5% CO₂. The seededcell count was 1×10⁴ cells/200 μL/well (except NanoCulture Plate) or1×10⁴ cells/250 μL/well (NanoCulture Plate), and the day of seeding wasdetermined to be Day 0. A half of medium exchange was performed asappropriate.

FIG. 1 shows the results. Cell morphology was confirmed by aphase-contrast microscope. It was determined whether or not a cell masswas formed based on the morphology, and in a case in whichspindle-shaped cells were observed on the bottom of the plate, it wasdetermined that there was adhesion of fibroblasts. As a result, in anyof PrimeSurface, Coming ULA Round-bottom, Corning ULA Flat-bottom, orElplasia, which is a three-dimensional culture plate as an ordinarylow-adhesive plate, adhesion of fibroblasts to the plate bottom was notobserved, and it was confirmed that a cell mass of primary cancer cellshaving a size of 100 μm or more could be formed, Meanwhile, in the96-well microplate (Coming Incorporated), which is an ordinary adhesiveplane culture plate, or NanoCulture Plate having an uneven scaffoldingstructure for cell mass formation on a low-adhesive plate, cell massformation was observed while adhesion of fibroblasts to the plate bottomwas also observed. The above results indicate that by performing cultureusing a low-adhesive culture plate and a medium containing a sol of anextracellular matrix, it is possible to allow a cell mass of primarycancer cells having a sufficient size to form without expansion orproliferation of cells such as fibroblasts other than cancer cells evenin suspension culture.

Example 3: Examination of Concentration of Extracellular Matrix to BeAdded to Medium

Primary cancer cells were prepared using pancreatic cancer (1) PDX tumor(procured from National Institutes of Biomedical Innovation, Health andNutrition) in accordance with Example 1. The necessary amount of cellscounted after dispersion treatment were collected in a 15-mL tube, andthe supernatant was removed by centrifugation at 300×g for 5 min.Thereafter, a medium prepared by adding Coming Matrigel GFR (CorningIncorporated) to StemPro hESC SFM (Thermo Fisher Scientific K.K.) toyield a final concentration of 0%, 0.5%, 1%, 2%, 5%, 10%, 20%, or 50%v/v was used for preparing cell suspension so that the cell count was5×10⁴ cells/mL. The cell suspension in an amount of 200 μL was seeded onPrimeSurface (Sumitomo Bakelite Co., Ltd.), and static culture wasinitiated in a CO₂ incubator set to 37° C., and 5% CO₂. The seeded cellcount was 1×10⁴ cells/200 μL/well, and the day of seeding was determinedto be Day 0. No medium exchange was performed, and the enablement of ahalf of medium exchange was evaluated on Day 14. FIG. 2 shows theresults. Cell morphology was confirmed by a phase-contrast microscope.Formation of a cell mass of primary cancer cells having a sufficientsize even in suspension was observed without expansion or proliferationof cells other than cancer cells such as fibroblasts at a ComingMatrigel GFR concentration of from 0.5% v/v to 5% v/v. In a case inwhich when half of the medium in each well was suctioned by pipetting,the medium was not sufficiently in a sol state, and thus, cells or cellmass was suctioned together, it was decided not to perform a half ofmedium exchange. It was possible to perform a half of medium exchange at2% v/v or less. It was found that the cell mass could be easily handledand used in various tests.

Example 4: Examination of Type of Extracellular Matrix to Be Added toMedium

Primary cancer cells were prepared using PDX tumor of pancreatic cancer(1) (procured from National Institutes of Biomedical Innovation, Healthand Nutrition) in accordance with Example 1. The necessary amount ofcells counted after dispersion treatment were collected in a 15-mL tube,and the supernatant was removed by centrifugation at 300×g for 5 min.Thereafter, a medium prepared by adding Coming Matrigel (CorningIncorporated), Cultrex BME (Trevigen, Inc.), Cultrex RGF BME (Trevigen,Inc.), or Cellmatrix Type I-A (Nitta Gelatin Inc.) to StemPro hESC SFM(Thermo Fisher Scientific K.K.) to yield a final concentration of 0%,0.5%, 1%, 2%, 5%, 10%, or 20% v/v was used for preparing cell suspensionso that the cell count was 5×10⁴ cells/mL. The cell suspension in anamount of 200 μL was seeded on PrimeSurface (Sumitomo Bakelite Co.,Ltd.), and static culture was initiated in a CO₂ incubator set to 37° C.and 5% CO₂. The seeded cell count was 1×10⁴ cells/200 μL/well, and theday of screening was determined to be Day 0. No medium exchange wasperformed, and the enablement of a half of medium exchange was evaluatedon Day 14. FIGS. 3-1 to 3-4 show the results. Cell morphology wasconfirmed visually or by a phase-contrast microscope. Formation of acell mass of primary cancer cells having a sufficient size even insuspension was observed without expansion or proliferation of cellsother than cancer cells such as fibroblasts at a concentration of anextracellular matrix of any of Coming Matrigel, Cultrex BME, Cultrex RGFBME, or Cellmatrix Type I-A from 0.5% v/v to 5% v/v. In a case in whichwhen half of the medium in each well was suctioned by pipetting, themedium was not sufficiently in a sol state, and thus, cells or cell masswas suctioned together, it was decided not to perform a half of mediumexchange. A half of medium exchange depended on the type of theextracellular matrix, and it was found possible to perform a half ofmedium exchange at from 2% to 5% v/v.

Example 5: Examination of Type of Minimal Essential Medium

Primary cancer cells were prepared using PDX tumor of pancreatic cancer(1) (procured from National Institutes of Biomedical Innovation, Healthand Nutrition) in accordance with Example 1. After dispersion treatment,the necessary amount of cells counted were collected in a 15-mL tube,and the supernatant was removed by centrifugation at 300×g for 5 min.Thereafter, a cell suspension was prepared so that the cell count was5×10⁴ cells/mL using a medium prepared by adding Corning Matrigel GFR(Coming Incorporated) to StemPro hESC SFM (Thermo Fisher ScientificK.K.) or Stemfit AK02N (Takara Bio Inc.) serving as a minimal essentialmedium to yield a final concentration of 2% v/v. The cell suspension inan amount of 200 μL was seeded on PrimeSurface (Sumitomo Bakelite Co.,Ltd.), and static culture was initiated in a CO₂ incubator set to 37° C.and 5% CO₂. The seeded cell count was 1×10⁴ cells/200 μL/well, and theday of seeding was determined to be Day 0. A half of medium exchange wasperformed as appropriate. FIG. 4 shows the results. Cell morphology wasconfirmed by a phase-contrast microscope. Formation of a cell mass ofprimary cancer cells having a sufficient size even in suspension wasobserved in the minimal essential medium of either StemPro hESC SFM orStemFit AK02N without expansion or proliferation of cells other thancancer cells such as fibroblasts.

Example 6: Examination of Type of PDX Tumor

Primary cancer cells were prepared using various PDX tumors (procuredfrom National Institutes of Biomedical Innovation, Health and Nutrition)in accordance with Example 1. As various PDX tumors, pancreatic cancer(1), pancreatic cancer (2), lung squamous cell carcinoma (1), lungsquamous cell carcinoma (2), lung squamous cell carcinoma (3), gastriccancer (1), and large bowel cancer (1) were used. After dispersiontreatment, the necessary amount of cells counted were collected in a15-mL tube, and the supernatant was removed by centrifugation at 300×gfor 5 min. Thereafter, a cell suspension was prepared so that the cellcount was 5×10⁴ cells/mL using a medium prepared by adding ComingMatrigel GFR (Coming incorporated) to StemPro hESC SFM (Thermo FisherScientific K K) to yield a final concentration of 2% v/v. The cellsuspension in an amount of 200 μL was seeded on PrimeSurface (SumitomoBakelite Co., Ltd.), and static culture was initiated in a CO₂ incubatorset to 37° C. and 5% CO₂. The seeded cell count was 1×10⁴ cells/200μL/well, and the day of seeding was determined to he Day 0. A half ofmedium exchange was performed as appropriate. FIG. 5 shows the results.Cell morphology was confirmed by a phase-contrast microscope. Formationof a cell mass of primary cancer cells having a sufficient size even insuspension was observed regardless of cancer species or type ofpatient-derived PDX tumor without expansion or proliferation of cellsother than cancer cells such as fibroblasts.

Example 7: Histopathological Analysis

Primary cancer cells were prepared using PDX tumor of pancreatic cancer(1) (procured from National Institutes of Biomedical Innovation. Healthand Nutrition) in accordance with Example 1. After dispersion treatment,the necessary amount of cells counted were collected in a 15-mL tube,and the supernatant was removed by centrifugation at 300×g for 5 min.Thereafter, a cell suspension was prepared so that the cell count was5×10⁴ cells/mi, using a medium prepared by adding Coming Matrigel GFR(Corning Incorporated) to StemPro hESC SFM (Thermo Fisher ScientificK.K.) to yield a final concentration of 2% v/v. The cell suspension inan amount of 200 μL it was seeded on PrimeSurface (Sumitomo BakeliteCo., Ltd.), and static culture was initiated in a CO₂ incubator set to37° C. and 5% CO₂. The seeded cell count was 1×10⁴ cells/200 μL/well,and the day of seeding was determined to be Day 0. A half of mediumexchange was performed as appropriate. Cell mass was collected in a15-mL tube on Day 14, solidified using iPGell (GENOSTAFF CO., LTD.) toform jelly, and fixed with a 10% neutral buffered formalin solutionovernight. In accordance with an ordinary method, a paraffin-embeddedspecimen was prepared using the formalin-fixed cell mass, and HEstaining and anti-human HLA imniunohistochemical staining wereperformed. As a control for comparison, a formalin-fixedparaffin-embedded specimen of a PDX tumor of pancreatic cancer (1) wasprepared, and HE staining and anti-human HLA immunohistochemicalstaining were performed in the same manner. FIG. 6 shows the results.The staining results confirmed that a cell mass formed from the PDXtumor of pancreatic cancer (1) was composed of human cancer cells. Itwas also confirmed that the PDX tumor of pancreatic cancer (1) and thecell mass formed therefrom had a similar structure.

Example 8: Examination of Cell Proliferation

Primary cancer cells were prepared using PDX tumor of pancreatic cancer(1) (procured from National Institutes of Biomedical Innovation, Healthand Nutrition) in accordance with Example 1. After dispersion treatment,the necessary amount of cells counted were collected in a 15-mL tube,and the supernatant was removed by centrifugation at 300×g for 5 min.Thereafter, a cell suspension was prepared so that the cell count was5×10⁴ cells/mL using a medium prepared by adding Coming Matrigel GFR(Coating Incorporated) to StemPro hESC SFM (Thermo Fisher ScientificK.K.) to yield a final concentration of 2% v/v. The cell suspension inan amount of 200 μL was seeded at N=4 (at 4 sites under the sameconditions) on PrimeSurface (Sumitomo Bakelite Co., Ltd.), and staticculture was initiated in a CO₂ incubator set to 37° C. and 5% CO₂. Theseeded cell count was 1×10⁴ cells/200 μL/well, and the day of seedingwas determined to be Day 0. A half of medium exchange was performed onDays 1, 7, 10, and 12. ATP assay was performed using CellTiter-Glo 3DViability Assay (Promega Corporation) on Days 1, 3, 7, 10, and 14. Theproportion of the result of each measurement day with respect to theresult of Day 1 was calculated, and a proliferation curve was created.FIG. 7 shows the results. The viable cell count of the cell massproduced from the PDX tumor of pancreatic cancer (1) increased in atime-dependent manner. Compared to Day 1, it increased linearly about 4times on Day 7 and 7 times or more on Day 14. This suggested that thecell mass had high proliferative ability.

Example 9: Anticancer Drug Sensitivity Test

Primary cancer cells were prepared using pancreatic cancer (1) PDX tumor(procured from National Institutes of Biomedical Innovation, Health andNutrition) in accordance with Example 1. An in vivo anticancer drugsensitivity test was performed by the following procedures withreference to a known method. A PDX tumor of pancreatic cancer (1), whichwas subcutaneously grown in an immunodeficient mouse [super SCID mouse(strain name: C3HeJ/NOs-scid; LPS-nonresponder)], was asepticallyextracted in a safety cabinet, and every necrotic area of the tumor wasremoved with surgical scissors. Subsequently, graft tumor fragments eachhaving a size of about 2 to 3 mm square were prepared and subcutaneouslygrafted in at least 12 immunodeficient mice. When the average tumorvolume reached about 200 mm³, the mice were grouped by tumor volume andassigned to a control group and a gemcitabine administration group (N=6:6 mice each under the same conditions). Gemcitabine (60 mg/kg/time) wasadministered at a dosing frequency of twice weekly for 4 weeks. Thetumor diameter was measured at a frequency of twice weekly and the tumorvolume was calculated, thereby creating a tumor proliferation curve.FIG. 8 (left) shows the results.

An in vitro anticancer drug sensitivity test was performed by thefollowing procedures. After dispersion treatment, the necessary amountof cells counted were collected in a 15-mL tube, and the supernatant wasremoved by centrifugation at 300×g for 5 min. Thereafter, a cellsuspension was prepared so that the cell count was 5×10⁴ cells/mL usinga medium prepared by adding Corning Matrigel GFR (Corning Incorporated)to StemPro hESC SFM (Thermo Fisher Scientific K.K.) to yield a finalconcentration of 2% v/v. The cell suspension in an amount of 200 μL wasseeded at N=4 (at 4 sites under the same conditions) on PrimeSurface(Sumitomo Bakelite Co., Ltd.), and static culture was initiated in a CO₂incubator set to 37° C. and 5% CO₂. The seeded cell count was 1×10⁴cells/200 μL/well, and the day of seeding was determined to be Day 0. Ahalf of medium exchange was performed on Day 1. On Day 7, a half ofmedium exchange was performed using a medium to which gemcitabine wasadded to yield a final gemcitabine concentration of 0, 0.001, 0.01, 0.1,1, or 10 μmol/L. On Days 10 and 12, a half of medium exchange wasperformed using a medium which was adjusted to have a stable finalgemcitabine concentration. ATP assay was performed using CellTiter-Glo3D Cell Viability Assay (Promega Corporation) on Day 14. The proportionof the result of each gemcitabine concentration group was calculatedwhen the result at a gemcitabine concentration of 0 μmol/L was definedas a cell viability of 100%, thereby creating a cell viability curve.FIG. 8 (right) shows the results.

Gemcitabine inhibited proliferation of the PDX tumor of pancreaticcancer (1) in both the in vivo and in vitro sensitivity tests.

Example 10: Examination Using Frozen Tumor Tissue

The PDX tumor of pancreatic cancer (1) (procured from NationalInstitutes of Biomedical Innovation, Health and Nutrition) was extractedin accordance with Example 1. The tumor was immersed in CELLBANKER 1(Takara Bio Inc.) and frozen by a Program Deep Freezer (Nepa Gene Co.,Ltd.), thereby preparing a frozen tumor. The frozen tumor was thawed ina warm bath at 37° C. and washed with HBSS (Thermo Fisher ScientificK.K.), and then, primary cancer cells were prepared in accordance withExample 1. After dispersion treatment, the necessary amount of cellscounted were collected in a 15-mL tube, and the supernatant was removedby centrifugation at 300×g for 5 min. Thereafter, a cell suspension wasprepared so that the cell count was 5×10⁴ cells/mL using a mediumprepared by adding Coming Matrigel GFR (Coming incorporated) to StemProhESC SFM (Thermo Fisher Scientific K.K.) to yield a final concentrationof 2% v/v. The cell suspension in an amount of 200 μL was seeded at N=4(at 4 sites under the same conditions) on PrimeSurface (SumitomoBakelite Co., Ltd.), and static culture was initiated in a CO₂ incubatorset to 37° C. and 5% CO₂. The seeded cell count was 1×10⁴ cells/200μL/well, and the day of seeding was determined to be Day 0. On Day 3, 50μL of a medium, to which gemcitabine was added to yield a finalgemcitabine concentration of 0, 0,001, 0.01, 0.1, 1, or 10 μmol/L, wasadded to each well (250 μL/well). ATP assay was performed usingCellTiter-Glo 3D Cell Viability Assay (Promega Corporation) on Days 0,3, and 7. The proportion of the result of each measurement day withrespect to the result of Day 0 was calculated, and a proliferation curvewas created. The proportion based on the result of each gemcitabineconcentration group was calculated when the result at a gemcitabineconcentration of 0 μmol/L was defined as a cell viability of 100%,thereby creating a cell viability curve. FIG. 9 shows the results.

The viable cell count of the cell mass produced from the once-frozen PDXtumor of pancreatic cancer (1) increased in a time-dependent manner.Compared to Day 0, it increased about 4 times on Day 7. As in Example 9,gemcitabine inhibited proliferation of a cell mass originating from thePDX tumor of pancreatic cancer (1) even with the use of a once-frozentumor tissue.

Example 11: Examination of Subculture

A fresh tumor-derived cell mass and a frozen tumor-derived cell masswere obtained in accordance with Examples 1 and 10, respectively, usinga PIX tumor of pancreatic cancer (1) (procured from National Institutesof Biomedical Innovation, Health and Nutrition). Each cell mass wascollected in a 50-mL tube, and the supernatant was removed bycentrifugation at 300×g for 5 min. Thereafter, the cell mass wassuspended in TrypLE (Thermo Fisher Scientific K.K.) and enzymaticallytreated in a warm bath at 37° C. The reaction was weakened by adding 10times the amount of HBSS (Thermo Fisher Scientific K.K.) to the reactionsolution. The undispersed residues were removed by passing the mixturethrough a 100-μm cell strainer. The tube and the cell strainer werewashed with an appropriate amount of HBSS, and cells were collected andcentrifuged at 300×g for 5 min. The supernatant was removed. Then, HBSSwas added to resuspend the resulting cell pellet and centrifuged at300×g for 5 min. Thereafter, the cell pellet was resuspended with anappropriate amount of HBSS and cell counting was performed. Afterconfirming that the cells were separated into single cells, the cellswere used in the following experiment.

The necessary amount of cells were collected in a 15-mL tube, and thesupernatant was removed by centrifugation at 300×g for 5 min.Thereafter, a cell suspension was prepared so that the cell count was5×10⁴ cells/mL (fresh tumor-derived) or 2×10⁴ cells/mL (frozentumor-derived) using a medium prepared by adding Coming Matrigel GFR(Corning Incorporated) to StemPro hESC SEM (Thermo Fisher ScientificK.K.) to yield a final concentration of 2% v/v. The cell suspension inan amount of 200 μL was seeded at N=4 (at 4 sites under the sameconditions) on PrimeSurface (Sumitomo Bakelite Co., Ltd.), and staticculture was initiated in a CO₂ incubator set to 37° C. and 5% CO₂. Theseeded cell count was 1×10⁴ cells/200 μL/well (fresh tumor-derived) or4×10³ cells/200 μL/well (frozen tumor-derived), and the day of seedingwas determined to be Day 0. On Day 3, 50 μL of a medium, to whichgemcitabine was added to yield a final gemcitabine concentration of 0,0.001, 0.01, 0.1, 1, or 10 μmol/L, was added to each well (250 μL/well).ATP assay was performed using CeWriter-Glo 3D Cell Viability Assay(Promega Corporation) on Days 0, 3, and 7. The ratio of the result ofeach measurement day with respect to the result of Day 0 was calculated,and a growth curve was created. The proportion based on the result ofeach gemcitabine concentration group was calculated when the result at agemcitabine concentration of 0 μmol/L was defined as a cell viability of100%, thereby creating a cell viability curve. FIGS. 10-1 and 10-2 showthe results.

The viable cell count of the cell mass reproduced (also referred to as“subcultured”) from the PDX tumor of pancreatic cancer (1) increased ina time-dependent manner regardless whether it was a fresh tumor-derivedor frozen tumor-derived cell mass. Compared to Day 0, it increasedlinearly about 2 times on Day 7. As in Examples 9 and 10, gemcitabineinhibited proliferation of the reproduced cell mass originating from thePDX tumor of pancreatic cancer (1).

INDUSTRIAL APPLICABILITY

According to the invention, it has become possible to provide a methodof producing a cell mass by three-dimensional culture of primary cancercells having proliferative ability and properties of handleability,versatility, and high-throughput performance, in which a human tumortissue is used as a starting material, proliferation of cells other thancancer cells such as fibroblasts is inhibited, and the cell massincludes primary cancer cells as a main component. Therefore, it becomespossible to easily and inexpensively produce a cell mass from an in vivotissue, which can contribute to drug screening, drug efficacyevaluation, drug safety evaluation, regenerative medicine, and the like.

1. A method of producing a cell mass by three-dimensional culture ofprimary cancer cells using a tumor tissue, comprising: athree-dimensional culture step of culturing cells obtained from thetumor tissue in a medium containing a 5% v/v or less extracellularmatrix on a substantially low-adhesive cell culture substrate.
 2. Themethod according to claim 1, wherein the tumor tissue is a xenografttumor.
 3. A cell mass of primary cancer cells obtained from a tumortissue, which is produced by the method of producing a cell massaccording to claim
 1. 4. A method of screening for a substance that actson a cell mass, comprising: producing a cell mass of primary cancercells by the method of producing a cell mass according to claim 1;adding a test substance to the cell mass; and evaluating an action ofthe test substance on the cell mass.
 5. A method of determining effectsof a substance on a cell mass, comprising: producing cell mass ofprimary cancer cells by the method of producing cell mass according toclaim 1; adding a test substance to the cell mass; and evaluatingeffects of the test substance on the cell mass.
 6. A kit of producing acell mass by three-dimensional culture of primary cancer cells using atumor tissue, comprising: a substantially low-adhesive cell culturesubstrate; and a medium containing a 5% v/v or less extracellular matrixfor a three-dimensional culture step.
 7. The method according to claim1, wherein said extracellular matrix is selected from the groupconsisting of collagen I, collagen IV, fibronectin, laminin,vitronectin, entactin, gelatin, elastin, proteoglycan,glucosaminoglycan, chondroitin sulfate, dermatan sulfate, heparansulfate, heparin, keratan sulfate, Matrigel (trademark: CorningIncorporated), Matrigel GFR (trademark: Corning Incorporated), CultrexBME (trademark: Trevigen, Inc.), Cultrex RGF BME (trademark: Trevigen,Inc.), Cellmatrix Type I-A (trademark: Nitta Gelatin Inc.), and growthfactors.
 8. The method according to claim 1, wherein the cell massobtained by the method has an average diameter of 100 μm or more,preferably from 100 μm to 300 μm.
 9. The method according to claim 1,wherein the substantially low-adhesive cell culture substrate is aculture substrate having a hydrophilic surface or a cell culturesubstrate having a surface treated with a hydrophilic compound.
 10. Themethod according to claim 1, wherein said medium contains 0.1% v/v ormore, preferably 0.2% v/v or more, and more preferably 0.5% v/v or moreand 5% v/v or less extracellular matrix.
 11. The method according toclaim 1, wherein said three-dimensional culture step is performed from 2days to 30 days.